This invention relates to a method of preparing and crystallizing Fraction I protein directly from unpurified extracts of a variety of plant species.
With the cost and inefficiency involved in the production of animal protein for human consumption and with the steady growth of world population, and increased usage of protein resources, it is becoming more apparent that nonconventional sources must be tapped for the production of high quality proteins that meet industrial needs as well as the nutritional standards and requirements for both human and animal consumption. Within the last thirty or so years, as the need for new sources of high quality protein has become crucial, terrestrial and aquatic plants have been seriously investigated as possible protein sources. Leaves of all plants contain certain soluble proteins that are generally rich in the essential amino acids and thus offer a high potential for the increased production of high quality protein for human and animal dietary needs and at affordable prices.
It is well-known in the art that considerable amounts of protein can be recovered from forage crops without destroying or decreasing their value as fodder or silage, by suitably modifying and improving available processing techniques. Under most current agricultural practices, however, valuable proteins obtainable from plants and forage crops are discarded and thus wasted.
In recent times, however, some techniques have been developed for the production of plant proteins. Such known techniques do not involve separating or identifying the nature, type and quality of the resulting proteins. Thus, such protein-rich products are in a form that is generally not of the highest quality and therefor unsuitable for human consumption. One such simple procedure for the preparation of a protein from the leaves of alfalfa consists of obtaining therefrom a dark green non-protein fraction that may be dried and used as animal fodder, and a white, bland, protein-rich fraction which is insoluble. Due to its insolubility, the protein-rich fraction is particularly unsuitable for human and anminal consumption as a food additive or fortifier.
The most abundant major plant protein is Fraction 1 protein. Its major component has been identified as the enzyme ribulose 1,5-bisphosphate carboxylase (RuBisCO). Fraction 1 protein is widely distributed in nature and constitutes up to 50% of the soluble protein contained in leaves and approximately 20% of the total plant protein. The amino acid composition of Fraction 1 protein is well balanced in terms of the essential and non-essential amino acids, comparing favorably with soybean, casein and animal proteins. The amounts of the essential amino acids in Fraction 1 protein, with the exception of methionine, meet or even exceed standards established for human nutritional requirements. The protein also has potential utility in the medical field.
Before its usefulness and possible incorporation as a food supplement can be laboratory and field tested this major important protein must be isolated and purified in large quantifies. It is essential that the protein be in a relatively pure form. However, current purification techniques are not applicable to large-scale isolation of RuBisCO in a pure form. Most published procedures for purifying Fraction 1 protein and its major component, RuBisCO, generally use small quantities of leaf tissue and require fairly elaborate analytical techniques such as ultracentrifugation and chromatography or sucrose gradient fractionation. While these procedures are useful for obtaining quantites of RuBisCO on a small laboratory scale sufficient for structural and enzymatic studies, they are incapable of purifying large quantities of the enzyme. It is physically impossible to scale-up most of these analytical methods to isolate and purify the enzyme in relatively pure form for its effective utilization in human and animal diets.
RuBisCO has also been crystallized, on the same analytical scale, using pre-purified extracts of seven plant species consisting of spinach, alfalfa, tomato, potato, corn, cotton, and tobacco. Such known methods have not been successful in the preparation of RuBisCO on a large, manufacturing scale nor have they been demonstrated to be effective in purifying the enzyme from unpurified extracts of plant species.
Currently only the RuBisCO from tobacco plants can be crystallized from unpurified extracts. The procedural steps heretofore used in the preparation of the crystalline tobacco enzyme involve the steps of (1) breaking the chloroplasts in the presence of high concentrations of sodium chloride to release the enzyme, (2) heating the resulting solution to precipitate all other undesirable components, (3) removing the excess sodium chloride by gel filtration, and (4) allowing the protein crystals to form.
However, crystallization is a complex phenomena and the molecular forces which cause proteins in general to crystallize are poorly understood. Solubility and viscosity properties of proteins differ from one protein to another and depend on the primary, secondary, tertiary (and where applicable), quarternary structures of the specific proteins. The nature and concentration of electrolytes present in the medium, the pH temperature, and concentration of the protein are crucial factors which influence the crystallization process. The choice of the precipitant employed in initiating nucleation and crystallization of proteins also plays an important role.
The optimum concentrations of these various reactants which are effective for one protein may or may not be effective for other proteins, due to wide variations in their structures. In addition, proteins are very sensitive to heat and chemicals which can cause their denaturation. Each protein, or even the same protein derived from different species, may vary in their primary, secondary, and tertiary structures and generally must be studied separately and independently to determine their structural and chemcial properties.
Existing procedures for the crystallization of tobacco RuBisCO, while a significant improvement over other earlier techniques, suffers many of the aforementioned drawbacks and is of limited utility. For instance the process can only be used successfully with tobacco leaves that are no more than 3-4 months old, such leaves being unavailable under current agricultural practices. All other sources of RuBisCO have proven unsatisfactory to crystallization by this procedure.
The need for gel filtration and the inherent limitations in using this step severely limit the total quantity of material processed. Furthermore the tobacco RuBisCO crystals obtained in this manner are not stable to lyophilization, which produces a powder that is insoluble, precluding its use in many formulations and as a food additive. Since the crystals have not been found to be capable of being freeze-dried, the nutritional value of the crystals may be diminished because 70% of the volume is aqueous. Storage and transfer may also become a problem.
Thus, there is a continual need for a method of preparation and crystallization of Fraction 1 protein and RuBisCO in sufficiently large quantities to enable its utilization as a major protein source for animal and human consumption.